Functionality of membrane proteins overexpressed and purified from E. coli is highly dependent upon the strain

Scientific Reports
Khadija MathieuCédric Orelle

Abstract

Overexpression of correctly folded membrane proteins is a fundamental prerequisite for functional and structural studies. One of the most commonly used expression systems for the production of membrane proteins is Escherichia coli. While misfolded proteins typically aggregate and form inclusions bodies, membrane proteins that are addressed to the membrane and extractable by detergents are generally assumed to be properly folded. Accordingly, GFP fusion strategy is often used as a fluorescent proxy to monitor their expression and folding quality. Here we investigated the functionality of two different multidrug ABC transporters, the homodimer BmrA from Bacillus subtilis and the heterodimer PatA/PatB from Streptococcus pneumoniae, when produced in several E. coli strains with T7 expression system. Strikingly, while strong expression in the membrane of several strains could be achieved, we observed drastic differences in the functionality of these proteins. Moreover, we observed a general trend in which mild detergents mainly extract the population of active transporters, whereas a harsher detergent like Fos-choline 12 could solubilize transporters irrespective of their functionality. Our results suggest that the amount of T7 RNA ...Continue Reading

References

Jan 1, 1992·Journal of Bacteriology·I IostM Dreyfus
Feb 7, 1998·European Journal of Biochemistry·A B ShapiroV Ling
Jul 4, 2001·The Journal of Biological Chemistry·R M Garavito, S Ferguson-Miller
Aug 10, 2001·Current Opinion in Structural Biology·J U Bowie
Aug 6, 2004·Protein Expression and Purification·Laurence Dumon-SeignovertLaurent Vuillard
Oct 20, 2004·Molecular Microbiology·J Gowrishankar, R Harinarayanan
Nov 3, 2004·Biochimica Et Biophysica Acta·Annela M SeddonPaula J Booth
May 26, 2005·Protein Expression and Purification·F William Studier
Dec 2, 2005·Nature·James U Bowie
Mar 24, 2006·Nature Methods·David DrewJan-Willem de Gier
Apr 21, 2007·Molecular & Cellular Proteomics : MCP·Samuel WagnerJan-Willem de Gier
Apr 9, 2008·Proceedings of the National Academy of Sciences of the United States of America·Eric R GeertsmaBert Poolman
Sep 18, 2008·Proceedings of the National Academy of Sciences of the United States of America·Samuel WagnerJan-Willem de Gier
Jan 24, 2009·Protein Science : a Publication of the Protein Society·Elizabeth Massey-GendelJames U Bowie
Jun 8, 2010·The Journal of Biological Chemistry·Shani LeviatanNathan Nelson
Jun 15, 2010·Journal of Molecular Biology·Daniel M LinaresBert Poolman
Aug 17, 2010·The Journal of Antimicrobial Chemotherapy·Farid El GarchFrançoise Van Bambeke
Feb 15, 2011·Journal of Molecular Biology·Mirjam M KlepschJan-Willem L de Gier
May 17, 2011·Microbial Cell Factories·Tomohiro MakinoGeorge Georgiou
Jul 2, 2011·Molecular & Cellular Proteomics : MCP·Francesca GubelliniJohn F Hunt
Sep 8, 2011·Proceedings of the National Academy of Sciences of the United States of America·Irina SmirnovaH Ronald Kaback
Dec 29, 2011·Biochimica Et Biophysica Acta·Joen LuirinkJan-Willem de Gier
Jun 20, 2012·Proceedings of the National Academy of Sciences of the United States of America·Shahid MehmoodJean-Michel Jault
Aug 4, 2012·Journal of Molecular Biology·Susan SchlegelJan-Willem de Gier
Sep 12, 2013·Journal of the American Chemical Society·Manuela ZoonensFrançois Dehez
Sep 18, 2013·Journal of Molecular Biology·Nadia GulBert Poolman
Nov 10, 2013·Biochimica Et Biophysica Acta·Susan SchlegelJan-Willem de Gier

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Citations

Aug 12, 2019·Microbial Cell Factories·Kerstin KanonenbergLutz Schmitt
Jun 30, 2019·Research in Microbiology·Cédric OrelleJean-Michel Jault
Oct 20, 2019·Fitoterapia·Fatma Gizem AvciBerna Sariyar Akbulut
Oct 9, 2021·Emerging Topics in Life Sciences·James Birch, Andrew Quigley

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Methods Mentioned

BETA
fluorescence microscopy
GTPase
size-exclusion chromatography
X-ray
PCR

Software Mentioned

ImageJ
MicrobeJ
NIS

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